The enzyme beta-D-glucuronidase catalyzes the hydrolysis of the O-glycosyl bond in beta-D-glucuronosides with the release of D-glucuronic acid. The enzyme is found in most vertebrates and many molluscs, but generally absent in higher plants, mosses, algae, ferns, fungi, as well as in most bacteria (U.S. Pat. No. 5,268,463).
The beta-D-glucuronidase is often used as a reporter for monitoring promoter activity (GUS reporter system) (U.S. Pat. No. 5,268,463). Indicators for detection of beta-D-glucuronidase consist of a dye molecule conjugated to glucuronic acid through a glycosidic bond, which can be cleaved by beta-D-glucuronidase. Cleavage liberates the dye which then unfolds its characteristic optical properties detectable by eye or suitable equipment. Common indicators in GUS reporter assays are X-beta-D-glucuronic acid, p-nitrophenyl-beta-D-glucuronic acid (PNPGluc) and 4-methylumbelliferyl-beta-D-glucuronide (MUG). However, the spectral properties of these indicators are not ideal. The yellow colour of p-nitrophenol, the cleavage product of PNPGluc, can be difficult to detect in a background with similar coloration, especially in plant tissues. Also, the blue fluorescence of 4-methylumbelliferone, the cleavage product of MUG, is often difficult to distinguish from natural matrix fluorescence (U.S. Pat. No. 5,268,463; U.S. Pat. No. 5,599,670).
Most importantly, beta-D-glucuronidase is a reliable marker for the presence and viability of Escherichia coli bacteria, therefore, providing the basis for common E. coli testing. Such test is typically conducted by contacting a suitable indicator substance sensitive to glucuronidase with a sample and observing the emergence of a signal associated with activity of glucuronidase. A popular fluorogenic indicator (an indicator producing fluorescence upon enzymatic transformation) for such use is MUG. Glucuronidase accepts this substance as a substrate and cleaves it hydrolytically—a process which is evident by the appearance of blue fluorescence.
In bacterial cultures the blue fluorescence of 4-methylumbelliferone is sometimes difficult to distinguish from natural matrix fluorescence or fluorescence from certain Pseudomonas strains (Manafi et al. (1991), Microbiol. Rev. vol. 55, pp. 335-348)). Superior both fluorogenic (e.g. fluorescein-, Resorufin-beta-D-glucuronic acid) and chromogenic (e.g. phenolphthalein-, sulfophenolphthalein-, Resorufin-beta-D-glucuronic acid) indicators are well known in the art but not commonly used due to their prohibitively high cost of manufacturing (U.S. Pat. No. 5,268,463; U.S. Pat. No. 5,599,670; U.S. Pat. No. 6,534,637).
Considering the relative simplicity of such molecules (beta-D-glucuronic acid glycosides) and the rapid advancement of synthetic organic chemistry over the past decades it is surprising that this problem has not yet been resolved. The key of the synthetic challenge is not associated with the formation of the glycosidic linkage between the dye and glucuronic acid as one might expect. It is the subsequent deprotection step of the glucuronic acid moiety of the indicator which causes the problem: For the course of synthesis the non-anomeric hydroxyl groups of glucuronic acid are chemically deactivated by acetylation and the carboxyl group is masked as an ester group. While the former can be removed with ease after glycosidation, cleavage of the glucuronic acid ester often leads to decomposition, rearrangement or lactonization of the indicator molecule. Until today, the lack of much needed sensitive and efficient indicators for glucuronidase is due to this one simple synthetic chemistry problem. Here we do not disclose the long-sought chemical solution to the problem but we describe our discovery that no such solution is needed.